An estimated 50-70% of catheter-related infections, 40-50% prosthetic cardiac-valve infections, and 20-50% of joint-replacement infections are caused by staphylococci biofilms, of which Staphylococcus aureus is the leading pathogen. The current challenges in biofilm studies are that the exact nature (composition) of the biofilm is difficult to determine, and the biofilm composition varies greatly from one organism to another. The characterization of heterogeneous biofilms is not amenable to conventional spectroscopic methods. In the next grant period, we propose to examine biofilms of S. aureus using combined solid-state NMR and liquid-chromatography/mass-spectrometry, to determine biofilm composition and local structure, and to establish the mode of action of drugs that can sterilize mature biofilms. We will gain insights into local structure and function in complicated biofilms by using a collection of specific stable-isotope labels. This development of solid-state NMR methods for in situ analyses of biofilms is a new platform for evaluating at the molecular level novel chemical and biological agents that target biofilms and hence holds promise for the treatment and prevention of biofilm-related infections. Our specific aims are: (i) Determine the composition and structural organization of mature biofilms formed by wild-type and mutant strains of S. aureus. (ii) Determine the mode of action of oritavancin in killing S. aureus cells under stationary-phase conditions. (iii) Determine the mode of action of novel anti-biofilm agents for S. aureus. The glycopeptide, oritavancin (which was approved for use in the clinic by the FDA in September, 2014) has been shown to kill stationary-phase biofilm S. aureus with a minimal biofilm eradication concentration of only 2-4 g/mL. No other known glycopeptide antibiotic has this sterilizing capability. We intend to focus on understanding the destruction of mature biofilms by oritavancin, with a secondary goal of unraveling its inhibition of biofilm formation. What we learn about biofilm inhibition and sterilization should be applicable to the design of second-generation oritavancin-like glycopeptide bactericides.